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Biomedical subjects

I Lefkovits

Publications and source records attributed to I Lefkovits.

At least 37 records · Page 2Linked to original sources
PubMed1995

How do B cells differ from T cells in terms of gene expression?

As part of an ongoing program to identify the genes that distinguish B cells from T cells, we have analyzed 910 molecular species of polypeptides detectable in mouse lymphocytes by 2D gel electrophoresis. Of these 910 polypeptides, 488 were present in both B and T cells, 185 in B but not T cells, and 237 in T but not B cells. The detected set of polypeptides accounts for more than 95% of the protein mass of lymphocytes. There are about 2000 other polypeptides that are below the threshold of detection. We have started to identify and to retrieve cDNA clones belonging to the B and T cell subset.

Animals↗
PubMed1995

Human lymphocyte cDNA ordered library analyzed by 2D gel electrophoresis. 1. Pooling strategy and matching of gel patterns.

We have analyzed an ordered library of 4,608 cDNA clones from the CEM human leukemic cell line. The aim was to facilitate gene retrieval, to enable immediate access to cDNA clones and to provide information on the protein expression of the individual clones in a 2D gel readout. The matrix array of 24 x 16 x 12, each position of which contained lambda jacII phage from one plaque, enabled us to establish pools of clones along the three axes (24 pools of complexity 192 cloned entities, 16 pools of complexity 288 and 12 pools of complexity 384). The total cDNA complexity is here reduced to such a level that spots which in more complex gels served as landmark spots are not present in each pool, and thus cannot serve as landmarks anymore. The image analysis of such gels and especially the matching of spots is not reliable under these circumstances. In order to achieve reliable matching, additional samples were created, such that pools were co-electrophoresed according to a special concatenation scheme; these samples then contained over-lapping elements (e.g., pools 1 + 2 + 3 and 3 + 4 + 5 have at least those spots in common, which originate from pool 3). This approach turned out to be feasible and we have completed the matching of one half of the ordered library. Already from the present stage of analysis we have obtained valuable information on the cDNA library and on the distribution of clones in this library.

DNA, Complementary↗
PubMed1995

Human lymphocyte cDNA ordered library analyzed by 2D gel electrophoresis. 2. Frequency distribution of mRNA populations.

Cell-free transcription and translation products from an ordered library of cDNA clones from the CEM human leukemic cell line were submitted to analysis using two-dimensional gel electrophoresis as a read out system. The matrix array of 24 x 16 x 12 wells contained in each of the positions lambda jacII phages from one plaque. Pools of clones along the three axes (24 x-pools, 16 y-pools and 12 z-pools) were established. Results obtained upon matching of 12 x-pools were scrutinized for estimating the frequency of cDNA molecular species in the library. The results obtained are interpreted in such a way that there are no discrete distributions of mRNA molecular species, but rather there is a continuous distribution of mRNA's covering a wide range of frequencies. The lowest frequency found was about 4.5 x 10(-4) and the highest 1.6 x 10(-2). About half of all clones can be found among these low frequency ones (each occurring 0.45 times among 1,000 clones).

Cell-Free System↗
PubMed1995

Human lymphocyte cDNA ordered library analyzed by 2D gel electrophoresis. 3. Analysis of individual clones.

We pursued the goal of obtaining global information on cDNA libraries from lymphocytes by combining data on the genes with data on the expressed proteins. Ordered libraries enabled us to establish the properties of the clones by direct retrieval of individual clones from the matrix (in our system a 24 x 16 x 12 array). We have picked-upon inspection of radiofluorographs obtained from the pools of the library--39 clones and performed a 2D gel analysis on the cell free expressed clonal products and have established also their partial nucleotide sequences. Details on two such clones are reported; clone CEM1001 is a new gene, while clone CEM1002 is a gene for human elongation factor 1-beta.

Base Sequence↗
PubMed1994

Data management in a two-dimensional gel electrophoresis laboratory: "gelscript 1" software.

We have developed a software package "gelscript 1", to perform data operations in a laboratory that runs and analyzes a large number of two-dimensional (2-D) gels. This system is managed automatically by the "4th Dimension" relational database on a Macintosh platform. A guiding principle in the development of "gelscript 1" was that any piece of information is entered into the system only once, and is retrieved, copied, inserted or checked whenever required. In addition to saving labor, this principle ensures consistency and integrity of the database. The system always "knows" how many gels were run from any one sample, how long the films were exposed or were supposed to be exposed. It keeps track of data throughout all operations, warns of improper sample assignments, and prints labels for films, samples, protocols, etc. The software can be adapted to other applications, in which many samples are processed and where cross-referencing is needed.

Databases, Factual↗
PubMed1994

The amino acid composition of 350 lymphocyte proteins.

We determined the amino acid composition of proteins of Sp2 hybridoma cells by a procedure which assembles the information on the polypeptides upon two-dimensional gel electrophoresis, such that biosynthetic labeling with 20 different 3H amino acids provides the data--spot intensities--on the relative representation of the detected polypeptides. The gels were impregnated with 2,5-diphenyloxazol (PPO) and suitably exposed radiofluorographs were selected for analysis. The images originating from the 12 cultures labeled with amino acids R, A, H, I, L, K, M, F, P, S, T and Y were analysed with the Kepler image analysis system. The spot volume data of the 12 analysed patterns were corrected for the unequal labeling efficiencies of the 3H amino acids and for the various exposure times. This correction is performed by applying calibration factors based on the amino acid determination of a hydrolysate of the analysed cells. After the calibration step was applied to the data files we used the amino acid compositions of nine proteins taken from a database to establish for each of these proteins the correlation coefficients with the image analysis derived amino acid compositions of all spots. The correlation coefficients allow us to tentatively identify polypeptide spots on two-dimensional gels, while the amino acid composition of 350 investigated two-dimensional gel spots is usable as an identification tag in the gene retrieval from our cDNA libraries.

Amino Acids↗
PubMed1994

Lymphocyte Proteinpaedia stage two: T-cell polypeptides from a partitioned cDNA library revealed by the dual decay method.

A complex population of gene products was analyzed by combining the great resolving power of two-dimensional (2D) protein electrophoresis with a detailed dissection of individual protein species afforded by cDNA cloning. A cDNA library from BW5147 was partitioned by random sampling into sectors (of a complexity of 500 phase plaques/sector). From each of the unique sets of cDNA clones present in the sector, 2D gels were prepared. Patterns of spots were analyzed using the Kepler software system, and the compiled data both from the sectors and from the natural lymphocyte populations have been established to serve as a guide for gene retrieval. Use of the dual decay method, which compares two exposures of a 2D gel co-electrophoretic pattern, obtained from samples with (35S)-polypeptides mixed with (3H)-polypeptides, is described and scrutinized. It was determined that of 268 spots (three sectors combined), 118 spots were shared with the natural lymphocyte population, the remaining 150 spots occur only in the sectors (and not detected in the cell population). All cDNA populations are available for retrieval. Information obtained throughout the work was entered into the database.

Animals↗
PubMed1994

From clones of cells to clones of genes.

Three approaches to study the immune system are presented. First is the limiting dilution analysis, second is the analysis of the polypeptide patterns of lymphocytes by two-dimensional gel electrophoresis, and the third is the utilization of a partitioned cDNA library for establishing a gene catalog and proteinpaedia. All the approaches were used to study clonal diversity and clonal heterogeneity. The gene catalog which is already established is available to recover genes expressed in various lymphocyte clones.

Animals↗
PubMed1993

Restriction sites as identification tags for the gene catalog: a 2D gel model.

In our effort to collect, organize and assemble data from lymphocyte cDNA libraries, we assign DNA restriction sites collectively to the spots on two-dimensional (2D) gel patterns. In order to test the efficiency and reliability of such an approach, we have modeled the restriction analysis of cDNA libraries with a panel of restriction endonucleases. The work has two parts. In the first, we have chosen 255 proteins from the EMBL data base and determined whether or not their coding sequences contain restriction sites for the enzymes of our choice. In order to apply a sufficient discriminatory power we decided to use a relatively large number of cleaving enzymes with low and high cutting frequencies. In total, 13 restriction enzymes were chosen, which could distinguish 2(13) or 8192 different restriction site combinations. We have compiled a table in which the absence or presence of restriction sites yields a pattern of 'zeros' and 'ones'. Such a restriction pattern can be read as a binary number. The binary numbers with maximally 13 digits would uniquely assign each of the 255 proteins if the nucleotide sequences would be truly at random. As the restriction sites are not randomly distributed, the 'typing' does not yield a unique assignment. The choice of sequences was not random either. In fact, there are some human nucleotide sequences which possess the same cut number (the decimal equivalent of the binary number representing the restriction pattern). In spite of this redundancy, 141 coding sequences could uniquely be distinguished by the above treatment. In the second part of the project we have used the above mentioned coding sequences to prepare two-dimensional maps (plots of charge vs size) of the same kind as one obtains from experimental 2D gels and submitted such a map together with 13 maps of restriction enzyme treated populations to a computer image analysis. Ideally, one would expect results (cut numbers) congruent to those obtained in the first part of the work. In the modeled system we were confronted with 2D maps which closely resembled the experimental situation (e.g. some spots were close together and overlapping) and instances of incorrect spot detection yielding 'false cut numbers'. From 255 proteins we were able to assign unequivocally 161 proteins. To implement the model in an actual experiment we will perform the digestion with the restriction enzymes in duplicate, and only spots assigned the same cut number upon the two independent treatments will be considered as carrying a valid restriction tag.

Base Sequence↗
PubMed1992

Putative invasion-specific proteins in mouse T-cell hybridomas that differ in invasive and metastatic potential.

Fusion of invasive, activated T-lymphocytes with non-invasive BW5147 T-lymphoma cells mainly yields highly invasive (HI), highly metastatic T-cell hybridomas. In addition, several non-invasive (NI), non-metastatic hybrids have been obtained, probably due to loss of involved gene(s) by chromosome segregation. Here we have compared a panel of HI and NI hybrids in a search for proteins specifically expressed by either cell type. MAbs were raised against HI hybrids, but out of more than 1,000 none bound exclusively to HI cells. Furthermore, polyclonal rat, rabbit and chicken antisera did not immunoprecipitate specific proteins from total lysates, and the expression of 18 (T-cell) surface markers did not correlate with invasiveness. These results indicated that the number of differences between HI and NI hybridomas was surprisingly small. This notion was confirmed by 2-dimensional gel electrophoresis. Among 1,000 detectable spots, we found only 2 clear-cut differences between HI and NI T-cell hybridomas, whereas multiple differences were found between individual hybrids. One protein (p130) was expressed at much higher levels by HI than by NI hybrids in this panel, whereas the other (p15) was only seen in NI hybrids. These proteins are primary candidates for a role in invasion.

Animals↗
PubMed1991

Light chain heterogeneity in the amphibian Xenopus.

Three subpopulations of light chains in Xenopus can be distinguished by monoclonal antibodies as well as by electrophoretic mobility on SDS-PAGE, peptide map and cell surface distribution. Analysis of these proteins from LPS-stimulated lymphocytes culture supernatants by two-dimensional gel electrophoresis showed a heterogeneity comparable to that observed for mouse kappa light chains. However, evidence from the selective expression of light chain subpopulations, as well as highly restricted light chain representation in anti-DNP antibodies, supports earlier findings that an antibody response in Xenopus is greatly limited in heterogeneity.

Animals↗
PubMed1990

A strategy for founding a global lymphocyte proteinpaedia and gene catalogue.

In this article, Ivan Lefkovits, John Kettman and Christopher Coleclough describe a strategy for a comprehensive analysis of the genes active in lymphocytes. It allows direct access to the coding sequence of any gene, provided that the two-dimensional gel spot coordinate position is known. The authors have constructed representative lymphocyte cDNA libraries and partitioned them into many sectors, each containing a unique set of cDNA clones. Data from the analysis of natural lymphocyte proteins is compiled into a 'proteinpaedia', which is matched to a gene catalogue comprising the partitioned recombinant phage collection.

Animals↗
PubMed1990

Molecular analysis of genetically determined target organ abnormalities in spontaneous autoimmune thyroiditis.

We have shown in earlier studies, that the development of spontaneous autoimmune thyroiditis (SAT) in chickens of the Obese strain (OS) depends on the presence of both, two dominant genes coding for an altered immune regulation and one recessive gene responsible for the susceptibility of the target organ for the autoimmune attack. The product(s) of the latter is (are) still not known. The present study was aimed at identifying possible candidates of cellular components of the thyroid gland of OS chicken and its SAT susceptible parental Cornell C-strain (CS) by high resolution 2-dimensional (2D) gel electrophoresis. For this purpose organ cultures of the thyroid, bursa, thymus and liver were established and the synthesized polypeptides were labelled by 35S-methionine. OS and CS organs were compared with those of healthy normal White Leghorn (NWL) controls. The autoradiographs of the 2D-gels obtained from individual samples after various labelling periods were subjected to comparative analysis. We have found both quantitative and qualitative differences of polypeptide spots between OS/CS and NWL organ samples, some of them specific for the thyroid gland. Although one has to be aware that in this multidimensional analytical approach numerous, still elusive pattern differences are revealed, the thyroid specific phenomena will be further scrutinized.

Animals↗
PubMed1990

Regulation of mRNA abundance in activated T lymphocytes: identification of mRNA species affected by the inhibition of protein synthesis.

Inhibition of protein synthesis has often been observed to increase the concentration of mRNAs that encode proteins associated with the regulation of cell division. As two-dimensional gel electrophoresis permits the simultaneous monitoring of individual elements in large populations of gene products, we have used this technique to assess the effect of cycloheximide treatment on the mRNA complement of activated mouse T cells in an objective fashion. Two-dimensional gels of proteins generated by cell-free translation of mRNA from T-cell blasts display about 400 spots; only 5 of these are reproducibly enhanced by cycloheximide treatment and about 4 are diminished. The cDNA cloning vector lambda jac allows analysis of large arrays of molecular clones by cell-free expression, and we have used it in a sibling selection scheme to isolate a clone of one of the prominently induced mRNA species, which we refer to as chx1. chx1 mRNA concentration is increased by cycloheximide treatment of activated B cells, as well as T cells, and it is rapidly and transiently induced, in a cycloheximide-enhanced manner, upon serum stimulation of resting 3T3 fibroblastoid cells. The chx1 protein is hydrophilic, is slightly basic, and has patches of homology with the Jun-D gene product. The chx1 gene is remarkable in its lack of detectable introns and of strong bias against CpG dinucleotides.

Amino Acid Sequence↗
PubMed1990

Polyoma-induced thymic epithelial tumors: analysis by 2D gel electrophoresis of tumors upon labeling the entire tumor bearing host.

Injection of neonatal C3H/Bi mice with polyoma virus (PTA-5) results after 4 to 20 weeks in the development of thymic epithelial tumors in 2/3 of the mice. These tumors appear to be the result of a neoplastic proliferation or transformation of thymic epithelial cells. The tumors contain immature lymphoid infiltrates characteristic of normal tissue and express the same function as normal tissue by providing the microenvironment for T cell maturation. To establish more comprehensively the relation of neoplastic to normal thymic epithelial cells, tumor-bearing mice and their normal counterparts were labeled in vivo by injection of S35-L-methionine. The stroma and lymphoid cells were separated and analyzed by two dimensional gel electrophoresis followed by radiofluorography. It was found that the epithelial portion of the tumor differed in several structural polypeptide components from normal tissue while the polypeptide composition of the thymocyte population remained unaltered. It was concluded that the neoplasia is confined to the epithelium compartment and the function of the neoplastic organ is maintained.

Animals↗
PubMed1989

Immunoglobulin diversification in bursal duct-ligated chickens.

The role of external antigen contact on immunoglobulin (Ig) diversification occurring in chicken bursal cells was evaluated. The entry of environmental antigens into the lumen of the bursa of Fabricius was prevented by ligating the bursal duct prior to hatching (BDL: bursal duct ligation). We used two-dimensional gel electrophoresis to compare the heterogeneity of Ig molecules from bursa cells of normal and BDL chickens. We have found that Ig diversity obtained from BDL chickens' bursae in two-dimensional gel analysis was similar to that of control birds. Furthermore, by using two monoclonal anti-idiotype antibodies to study intrabursal Ig diversification we have shown that frequencies of the Cld-1 and Cld-2 idiotypes were also unaltered following bursal ligation. We conclude that primary B cell diversification in the bursa is independent of the external antigen flow from the bursal lumen.

Age Factors↗
PubMed1989

Consecutive radiofluorography and silver staining of two-dimensional gel electrophoretograms: application in determining the biosynthesis of serum and tissue proteins.

The methodology for conventional radiofluorography of two-dimensional gels, followed by rehydration of the gel and subsequent silver staining, is described. The image obtained by radiofluorography is referred to as biosynthetic image, and the image obtained by silver staining as constitutive image. Since the two images are already in close register (the same gel), reliable identification of polypeptides by the two different assays is possible, and the comparison provides valuable information on the catabolism of each entity. The utility of this procedure is illustrated in experiments involving a labeling with L-[35S]methionine of an entire mouse. Both serum and tissue samples were analyzed by two-dimensional gel electrophoresis with the aim of determining several categories of polypeptides in terms of their biosynthetic rates and their composition.

Animals↗